Sialylated glycoprotein compositions and uses thereof

ABSTRACT

The present application relates to sialylated glycoprotein compositions and methods of their use in treating various conditions and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.Continuation of U.S. patent application Ser. No. 14/639,171, filed Mar.5, 2015, (now U.S. Pat. No. 9,457,067, issued Oct. 4, 2016), whichclaims priority to U.S. Provisional Application Ser. No. 61/948,421,filed Mar. 5, 2014, and U.S. Provisional Application Ser. No.62/114,313, filed Feb. 10, 2015, each of which is herein incorporated byreference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to sialylated glycoprotein compositionsand methods of their use in treating various conditions and disorders.

DESCRIPTION OF TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ULPI_020_02US_SeqList_ST25.txt, date recorded: Mar. 2, 2015, file size:9 kilobytes).

BACKGROUND OF THE INVENTION

The number of commercially-available therapeutic proteins has increaseddramatically in recent years and most of these proteins areglycoproteins. The presence of sialic acid in a glycoprotein canpositively affect absorption, serum half-life, and clearance from theserum, as well as the physical, chemical and immunogenic properties ofthe respective glycoprotein. In certain circumstances, it may thereforebe desirable to increase the sialic acid content of a glycoproteinintended for use in pharmacologic applications.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatrecombinant glycoprotein produced from mammalian cells through the useof serum/protein free media improves sialylation of the recombinantglycoprotein, e.g., without reducing the M6P content of the recombinantglycoprotein.

In some embodiments of the present invention, a composition comprises arecombinant glycoprotein having a sialic acid content greater than 0.05mol/mol of the recombinant glycoprotein. In some embodiments, acomposition comprises a recombinant glycoprotein having a sialic acidcontent greater than 0.1 mol/mol of the recombinant glycoprotein. Insome embodiments, a composition comprises a recombinant glycoproteinhaving a sialic acid content greater than 0.5 mol/mol of the recombinantglycoprotein. In some embodiments, a composition comprises a recombinantglycoprotein having a sialic acid content greater than 0.7 mol/mol ofthe recombinant glycoprotein. In some embodiments, a compositioncomprises a recombinant glycoprotein having a sialic acid contentgreater than 1 mol/mol of the recombinant glycoprotein. In someembodiments, a composition comprises a recombinant glycoprotein having asialic acid content greater than 1.5 mol/mol of the recombinantglycoprotein. In some embodiments, a composition comprises a recombinantglycoprotein having a sialic acid content greater than 2 mol/mol of therecombinant glycoprotein. In some embodiments, a composition comprises arecombinant glycoprotein having a sialic acid content greater than 5mol/mol of the recombinant glycoprotein. In some embodiments, acomposition comprises a recombinant glycoprotein having a sialic acidcontent greater than 10 mol/mol of the recombinant glycoprotein. In someembodiments, a composition comprises a recombinant glycoprotein having asialic acid content greater than 20 mol/mol of the recombinantglycoprotein.

In some embodiments, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of galactose residues of the recombinantglycoprotein are sialylated.

In some embodiments, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein the recombinantglycoprotein is human β-glucuronidase and has a sialylation content ofat least 0.7 mol/mol of the recombinant glycoprotein.

In some other embodiments, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein the recombinantglycoprotein is human β-glucuronidase and has a sialylation content ofat least 0.7 mol/mol of the recombinant glycoprotein and a high level ofmannose-6-phosphate (M6P) moieties.

In one embodiment, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein the recombinantglycoprotein is human β-glucuronidase and has a sialylation content ofat least 0.7 mol/mol of the recombinant glycoprotein and a high level ofmannose-6-phosphate (M6P) moieties of at least 10 mol % of the totalglycan of the recombinant glycoprotein.

In another embodiment, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein the recombinantglycoprotein is human β-glucuronidase and has a sialylation content ofat least 0.7 mol/mol of the recombinant glycoprotein and a high level ofmannose-6-phosphate (M6P) moieties, e.g., K uptake is at its 60%, 70%,80%, 90% or maximum such as from about 1 nM to about 3 nM when tested inhuman fibroblast cells (MPS7).

In yet another embodiment, the present invention provides a compositioncomprising a recombinant glycoprotein, wherein the recombinantglycoprotein is human β-glucuronidase and has a sialylation content ofat least 0.7 mol/mol of the recombinant glycoprotein and a high level ofmannose-6-phosphate (M6P) moieties, e.g., half-maximal uptake in humanfibroblast cells such as concentrations at which the glycoprotein (e.g.,human β-glucuronidase) reaches 50% of maximal uptake is about no morethan 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM.

In some embodiments, the present invention provides a preparation of apopulation of a recombinant glycoprotein, wherein at least 50 percent ofthe population is sialylated. In some embodiments, the present inventionprovides a preparation of a population of a recombinant glycoprotein,wherein at least 60 percent of the population is sialylated. In someembodiments, the present invention provides a preparation of apopulation of a recombinant glycoprotein, wherein at least 70 percent ofthe population is sialylated. In some embodiments, the present inventionprovides a preparation of a population of a recombinant glycoprotein,wherein at least 80 percent of the population is sialylated. In someembodiments, the present invention provides a preparation of apopulation of a recombinant glycoprotein, wherein at least 90 percent ofthe population is sialylated.

Also provided is a method of making a composition/preparation accordingto the present invention. In some embodiments, the method comprisesexpressing the recombinant glycoprotein in a cell culture with a serumor protein free media. In some embodiments, a protein-free, chemicallydefined media may be used to grow cells. In some embodiments, the mediado not include an effective amount of a sugar selected from galactose,fructose, n-acetyl-mannosamine, mannose and combinations thereof. Forexample, the effective amount of a sugar is greater than 0.01 mM, 0.05mM, or 0.1 mM.

In some embodiments, a cell culture comprises a mammalian cell.Exemplary mammalian cells include but are not limited to Chinese HamsterOvary (CHO), HeLa, VERO, BHK, Cos, MDCK, 293, 3T3, myeloma (e.g. NSO,NSI), or WI38 cells. In a specific embodiment, the mammalian cells areChinese Hamster Ovary (CHO) cells.

In some other embodiments, a cell culture comprises a plant cell.Exemplary plant cells include but are not limited to carrot cells or anyother plant cell based cell culture, e.g., developed for recombinantprotein production.

Further provided is a method of treating lysosomal storage disorder(LSD) comprising administering to an individual in need of suchtreatment a therapeutically effective amount of thecomposition/preparation as described herein. In an exemplary embodiment,the composition/preparation comprises recombinant human β-glucuronidase.In a further exemplary embodiment, the LSD is mucopolysaccharidosis type7 (i.e., MPS 7, MPS VII, or Sly Syndrome). In some embodiments, therecombinant human β-glucuronidases provided herein have increased sialicacid content and are particularly useful in treating a LSD, e.g., MPS 7.

In some embodiments, the present invention provides a method fortreating a lysosomal storage disorder (LSD) in a subject, comprisingadministering a regimen of the composition/preparation as describedherein, wherein the administration provides a statistically significanttherapeutic effect for the treatment of the LSD. In an exemplaryembodiment, the composition/preparation comprises recombinant humanβ-glucuronidase. In a further exemplary embodiment, the LSD is MPS 7.

Definitions

As used herein, the term “effective” (e.g., “an effective amount”) meansadequate to accomplish a desired, expected, or intended result. Aneffective amount can be a therapeutically effective amount. A“therapeutically effective amount” refers to the amount of an activeingredient that, when administered to a subject, is sufficient to effectsuch treatment of a particular disease or condition. The“therapeutically effective amount” will vary depending on, e.g., thedisease or condition, the severity of the disease or condition, and theage, weight, etc., of the subject to be treated.

In general, “treating” or “treatment” of any condition, disease ordisorder refers, in some embodiments, to ameliorating the condition,disease or disorder (i.e., arresting or reducing the development of thedisease or at least one of the clinical symptoms thereof). In someembodiments “treating” or “treatment” refers to ameliorating at leastone physical parameter, which may not be discernible by the subject. Insome embodiments, “treating” or “treatment” refers to inhibiting thecondition, disease or disorder, either physically, (e.g., stabilizationof a discernible symptom), physiologically, (e.g., stabilization of aphysical parameter) or both. In some embodiments, “treating” or“treatment” refers to delaying the onset of a condition, disease, ordisorder.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive. It is specifically contemplatedthat any listing of items using the term “or” means that any of thoselisted items may also be specifically excluded from the relatedembodiment.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

A glycoprotein, as used herein, is a protein that has been modified bythe addition of one or more carbohydrates, including, especially, theaddition of one or more sugar residues.

As used herein, “GUS” refers to β-glucuronidase, an exemplaryglycoprotein in accordance with the present invention.

Sialylation, as used herein, is the addition of a sialic acid residue toa protein, which may be a glycoprotein. The term sialic acid, as usedherein, encompasses a family of sugars containing 9 or more carbonatoms, including a carboxyl group. A generic structure encompassing allknown natural forms of sialic acid is shown below.

R1 groups at various positions on a single molecule can be the same asor different from each other. R1 can be a hydrogen or an acetyl, lactyl,methyl, sulfate, phosphate, anhydro, sialic acid, fucose, glucose, orgalactose group. R2 can be an N-acetyl, N-glycolyl, amino, hydroxyl,N-glycolyl-O-acetyl, or N-glycolyl-O-methyl group. R3 represents thepreceding sugar residue in an oligosaccharide to which sialic acid isattached in the context of a glycoprotein. R3 can be galactose(connected at its 3, 4, or 5 position), N-acetyl-galactose (connected atits 6 position), N-acetyl-glucose (connected at its 4 or 6 position),sialic acid (connected at its 8 or 9 position), or5-N-glycolyl-neuraminic acid. Essentials of Glycobiology, Ch. 15, Varkiet al., eds., Cold Spring Harbor Laboratory Press, New York (1999). Morethan 40 forms of sialic acid have been found in nature. Essentials ofGlycobiology, Ch. 15, Varki et al., eds., Cold Spring Harbor LaboratoryPress, New York (1999). A common form of sialic acid isN-acetylneuraminic acid (NANA), in which R1 is a hydrogen at allpositions and R2 is an N-acetyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot comparing pharmacokinetics of recombinant humanβ-glucuronidases, GUS CR01 vs. GUS Lot 43/44 in rats via a two-stageinfusion. The data show that the infusion with the higher sialylatedCR01 results in less rapid clearance and a higher mean concentrationduring the infusion, which increases the total exposure to the enzyme,potentially enhancing its penetration of tissues that are more difficultto treat.

FIG. 2 is a plot showing only the post infusion clearance phase of GUSCR01 vs. GUS Lot 43/44 which was used to calculate the t1/2 values.These differences in the rate of clearance are sufficient to result inhigher serum levels of enzymes as shown in FIG. 1.

FIG. 3 is a series of plots showing tissue GUS levels for thepharmacokinetics study of GUS CR01 vs. GUS Lot 43/44. The tissuedelivery and uptake of human β-glucuronidases is shown to be enhanced asthe total uptake of sialylated human β-glucuronidases (CR01) is higherin all tissues than the lower sialylated enzyme (43-44). When theendogenous glucuronidase activity is substracted, the effective deliveryof therapeutic human β-glucuronidases is increased 2 fold to almost 10fold, an exceptional and surprising finding.

FIG. 4 is a plot showing the measurement of urinary glycosaminoglycan(uGAG) levels over 36 weeks in three subjects treated with recombinanthuman β-glucuronidase (rhGUS). A rapid and sustained dose-dependentreduction in uGAG levels was observed in subjects treated with rhGUS.

FIG. 5 illustrates the mean reduction in urinary glycosaminoglycan(uGAG) levels at the end of each dosing interval during a 36 weekevaluation of three subjects treated with rhGUS. A 4 mg/kg QOW doseresulted in the greatest reduction in uGAG levels.

FIG. 6 is a plot showing the measurement of serum glycosaminoglycan(GAG) levels over 36 weeks in three subjects treated with rhGUS. Eachsubject demonstrated at least a 25% reduction in serum GAG levels at theend of the 36-week treatment schedule.

FIG. 7 is a plot showing the measurement of liver size in subjectstreated with rhGUS. A significant reduction in hepatomegaly resultingfrom the 36 week treatment protocol was observed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery thatrecombinant glycoprotein produced from mammalian cells through the useof serum/protein free media improves sialylation of the recombinantglycoprotein, and additionally levels of mannose-6-phosphate moieties ofthe recombinant glycoprotein.

Sialylated Glycoprotein Compositions

Compositions as described herein comprise one or more glycoproteins thathave a high level or increased sialic acid content.

Sialic acids represent a family of aminosugars with 9-carbons with over50 members derived from N-acetyleuraminic acid. Sialic acid is only onecomponent out of several monosaccharides building glycans ofglycoproteins, but has an outstanding impact on the quality andstability of any therapeutic glycoproteins for several reasons: (I)terminal galactose residues are one of the major factors determining theserum half-life of glycoproteins. The serum half-life is regulated bythe expression of liver asialo-glycoprotein receptors. These receptorsbind nonsialylated glycoproteins and bound asialo-glycoproteins areremoved from the serum by endocytosis. As a consequence, expression ofterminal sialic acid on galactose residues prevents serum glycoproteinsfrom degradation; (II) sialic acids are important for masking antigenicdeterminants or epitopes. It is known that the receptors of the immunesystem (T- and B-cell receptors) often prefer nonsialylated structures.Therefore, the possibility of the generation of antibodies against thetherapeutic glycoproteins correlates with the degree of its sialylation;(III) negatively charged sialic acids influence protein-specificparameters such as the thermal stability, the resistance to proteolyticdegradation or its solubility (Bork et al., Increasing the Sialylationof Therapeutic Glycoproteins: The potential of the Sialic AcidBiosynthetic Pathway, Journal of Pharmaceutical Sciences, Vol. 98, No.10, October 2009).

In one aspect, the invention provides compositions comprising arecombinant glycoprotein having a sialic acid content greater than 0.05mol/mol, 0.1 mol/mol, 0.5 mol/mol, 0.7 mol/mol, 1 mol/mol, 1.5 mol/mol,2 mol/mol, 5 mol/mol, 10 mol/mol or 20 mol/mol of the recombinantglycoprotein. In some embodiments, the invention provides compositionscomprising a recombinant glycoprotein having a sialic acid contentgreater than 0.5 mol/mol of the recombinant glycoprotein. In additionalembodiments, the invention provides compositions comprising arecombinant glycoprotein having a sialic acid content greater than 0.7mol/mol of the recombinant glycoprotein. In certain additionalembodiments, the invention provides compositions comprising arecombinant glycoprotein having a sialic acid content greater than 1mol/mol of the recombinant glycoprotein.

In certain embodiments, the recombinant glycoprotein is a recombinantform of human β-glucuronidase, an enzyme responsible for catalyzing thehydrolysis of β-D-glucuronic acid residues from the non-reducing end ofmucopolysaccharides. In some embodiments, the recombinant humanβ-glucuronidase (rhGUS) has a sialic acid content greater than 0.1mol/mol, 0.5 mol/mol, 0.7 mol/mol, 1 mol/mol, 1.5 mol/mol, 2 mol/mol, or5 mol/mol of the rhGUS. In one exemplary embodiment, the recombinanthuman β-glucuronidase (rhGUS) has a sialic acid content greater than 0.7mol/mol of the rhGUS. In another exemplary embodiment, the recombinanthuman β-glucuronidase (rhGUS) has a sialic acid content greater than 1.0mol/mol of the rhGUS. In yet another exemplary embodiment, therecombinant human β-glucuronidase (rhGUS) has a sialic acid content ofabout 1.2 mol/mol of the rhGUS.

In some embodiments, the recombinant human β-glucuronidase (rhGUS) has asialic acid content of about 0.5 mol/mol to about 2.0 mol/mol of therhGUS. In one embodiment, the recombinant human β-glucuronidase (rhGUS)has a sialic acid content of about 0.6 mol/mol to about 1.5 mol/mol ofthe rhGUS. In another embodiment, the recombinant human β-glucuronidase(rhGUS) has a sialic acid content of about 0.7 mol/mol to about 1.4mol/mol of the rhGUS. In an exemplary embodiment, the recombinant humanβ-glucuronidase (rhGUS) has a sialic acid content of about 0.8 mol/molto about 1.3 mol/mol of the rhGUS. In another exemplary embodiment, therecombinant human β-glucuronidase (rhGUS) has a sialic acid content ofabout 1.0 mol/mol to about 1.2 mol/mol of the rhGUS.

In some embodiments, the composition of the present invention includes arecombinant glycoprotein having at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90% of sites suitable for sialic acid linkage sialylated. Ingeneral, galactose is the site suitable for sialic acid linkage orsialylation. In certain embodiments, a composition comprises arecombinant glycoprotein, wherein at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the galactose residues of the recombinantglycoprotein are sialylated. In some embodiments, the compositioncomprises a recombinant glycoprotein, wherein at least 50% of thegalactose residues of the recombinant glycoprotein are sialylated. Inadditional embodiments, the composition comprises a recombinantglycoprotein, wherein at least 60% of the galactose residues of therecombinant glycoprotein are sialylated. In certain additionalembodiments, the composition comprises a recombinant glycoprotein,wherein at least 70% of the galactose residues of the recombinantglycoprotein are sialylated.

In certain embodiments, the recombinant glycoprotein is a recombinanthuman β-glucuronidase (rhGUS). In some embodiments, at least 40%, 50%,60%, 70%, or 80% of the galactose residues of the recombinant humanβ-glucuronidase (rhGUS) are sialylated. In one exemplary embodiment, atleast 50% of the galactose residues of the rhGUS are sialylated. Inanother exemplary embodiment, at least 60% of the galactose residues ofthe rhGUS are sialylated. In yet another exemplary embodiment, at least70% of the galactose residues of the rhGUS are sialylated. In yetanother exemplary embodiment, about 70%, 71%, 72%, 73%, 74%, or about75% of the galactose residues of the rhGUS are sialylated.

In some embodiments, at least about 40% to at least about 90% of thegalactose residues of the recombinant human β-glucuronidase (rhGUS) aresialylated. In one embodiment, at least about 50% to at least about 80%of the galactose residues of the recombinant human β-glucuronidase(rhGUS) are sialylated. In another embodiment, at least about 60% to atleast about 80% of the galactose residues of the recombinant humanβ-glucuronidase (rhGUS) are sialylated. In an exemplary embodiment, atleast about 65% to at least about 75% of the galactose residues of therecombinant human β-glucuronidase (rhGUS) are sialylated.

In another aspect, the invention provides compositions comprising arecombinant glycoprotein having a high level of sialic acid content aswell as a high level of mannose-6-phosphate (M6P) moieties. As usedherein, M6P moieties include any mannose-6-phosphate capable of bindingto or being recognized by M6P receptors including without any limitationmono-phosphorylated and bis-phosphorylated mannose-6-phosphate. In oneembodiment, M6P moieties include any M6P binding to cation-independentM6P receptor (CI-MPR). In another embodiment, M6P moieties include anyM6P binding to cation-dependent M6P receptor (CD-MRP). In yet anotherembodiment, M6P moieties include any bis-phosphorylated M6P.

According to the present invention, a high level of mannose-6-phosphatemoieties can include any level of M6P moieties that is considered highby one skilled in the art, e.g., measured using any suitable means knownto or later developed by one skilled in the art. In one embodiment, ahigh level of M6P moieties of a recombinant glycoprotein includes M6Pmoiety levels of at least 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol% or 15 mol % of the total glycan of the recombinant glycoprotein. Forexample, the recombinant glycoprotein can have a high level of M6P asdetermined by the percentage of M6P peak area over total glycan peakarea, e.g., at least 10%, 11%, 12%, 13%, 14% or 15%. In someembodiments, the recombinant glycoprotein is a recombinant humanβ-glucuronidase (rhGUS) and comprises M6P moiety levels of at least 10mol %, 11 mol %, 12mol %, 13 mol %, 14 mol % or 15 mol % of the totalglycan of the rhGUS. In an exemplary embodiment, the rhGUS comprises M6Pmoiety levels of about 13% to about 15%.

In another embodiment, a high level of M6P moieties of a recombinantglycoprotein includes a high level of uptake of the recombinantglycoprotein by human cells, e.g., high affinity uptake amount by humanfibroblast cells. For example, the recombinant glycoprotein can have aM6P dependent K uptake of no more than 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2 nM, 2.1 nM, 2.2 nM, 2.3nM, 2.4 nM, 2.5 nM, 2.6 nM, 2.7 nM, 2.8 nM, 2.9 nM, 3 nM, 4 nM, or 5 nMby any suitable human cells, e.g., human fibroblast cells. In someembodiments, the recombinant glycoprotein is a recombinant humanβ-glucuronidase (rhGUS) and has a M6P dependent K uptake of less than 5nM, less than 4 nM, less than 3 nM, or less than 2 nM. In an exemplaryembodiment, the rhGUS has a M6P dependent K uptake of about 1.2 nM toabout 1.8 nM.

In yet another embodiment, a high level of M6P moieties in a recombinantglycoprotein includes lower concentrations required to achieve maximumuptake of the recombinant glycoprotein by human cells, e.g., lowerhalf-maximum concentration. For example, the recombinant glycoproteincan achieve maximum uptake by human cells, e.g., fibroblast cells atconcentrations less than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3nM, 2 nM or 1 nM. In some embodiments, the recombinant glycoprotein is arecombinant human β-glucuronidase (rhGUS) can achieve maximum uptake byhuman cells, e.g., fibroblast cells at concentrations of less than 5 nM,less than 4 nM, less than 3 nM, or less than 2 nM. In an exemplaryembodiment, the rhGUS can achieve maximum uptake by human cells, e.g.,fibroblast cells at concentrations of about 1.2 nM to about 1.8 nM.

In still another embodiment, a high level of M6P moieties in arecombinant glycoprotein includes one or more levels of M6P moietiescorresponding to levels of M6P moieties associated with naturalsialylation content of the recombinant glycoprotein, e.g., sialylationcontent of the recombinant glycoprotein prior to any means forenhancement such as using the methods disclosed in the presentapplication.

According to the present invention, in some embodiments the recombinantglycoprotein, e.g., a recombinant human β-glucuronidase or any otherlysosomal enzyme, has a sialylation content of at least 1 mol/mol and ahigh level of M6P moieties of at least 10 mol %, 11 mol %, 12 mol %, 13mol %, 14 mol % or 15 mol % of the total glycan of the recombinantglycoprotein. In one embodiment, the recombinant glycoprotein, e.g., arecombinant human β-glucuronidase or any other lysosomal enzyme, has asialylation content of at least 1 mol/mol and a high level of M6Pmoieties with an uptake of at least 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM or 2 nM by human cells, e.g.,human fibroblast cells. In another embodiment, the recombinantglycoprotein, e.g., a recombinant human β-glucuronidase or any otherlysosomal enzyme has a sialylation content of at least 1 mol/mol and ahigh level of M6P moieties with maximum uptake by human cells, e.g.,human fibroblast cells at less than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM,4 nM, 3 nM, 2 nM, or 1 nM of the recombinant glycoprotein.

It yet another aspect, the invention provides a composition comprising apopulation of recombinant glycoproteins, wherein at least 50%, 60%, 70%,80%, or 90% of the population is sialylated. In one embodiment, at least50%, 60%, 70%, 80% or 90% of the population is recombinant glycoproteinin accordance to the present invention, e.g., with respect tosialylation content and M6P level.

The recombinant glycoprotein provided by the present invention can beany glycoprotein. Exemplary recombinant glycoproteins include thosecomprising amino acid sequences identical to or substantially similar toall or part of one of the following proteins: a Flt3 ligand (asdescribed in WO 94/28391), a CD40 ligand (as described in U.S. Pat. No.6,087,329), erythropoietin, thrombopoietin, calcitonin, leptin, IL-2,angiopoietin-2 (as described by Maisonpierre et al. (1997), Science277(5322):55-60, incorporated herein by reference), Fas ligand, ligandfor receptor activator of NF-kappa B (RANKL, as described in WO01/36637), tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL, as described in WO 97/01633), thymic stroma-derivedlymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor (GM-CSF, as describedin Australian Patent No. 588819), mast cell growth factor, stem cellgrowth factor (described in e.g. U.S. Pat. No. 6,204,363, incorporatedherein by reference), epidermal growth factor, keratinocyte growthfactor, megakaryote growth and development factor, RANTES, growthhormone, insulin, insulinotropin, insulin-like growth factors,parathyroid hormone, interferons including α interferons, γ interferons,and consensus interferons (such as those described in U.S. Pat. Nos.4,695,623 and 4,897,471, both of which are incorporated herein byreference), nerve growth factor, brain-derived neurotrophic factor,synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon,interleukins 1 through 18, colony stimulating factors, lymphotoxin-β,tumor necrosis factor (TNF), leukemia inhibitory factor, oncostatin-M,and various ligands for cell surface molecules ELK and Hek (such as theligands for eph-related kinases or LERKS). Descriptions of proteins thatcan be produced according to the inventive methods may be found in, forexample, Human Cytokines: Handbook for Basic and Clinical Research, Vol.II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, Mass.,1998); Growth Factors: A Practical Approach (McKay and Leigh, eds.,Oxford University Press Inc., New York, 1993); and The Cytokine Handbook(A. W. Thompson, ed., Academic Press, San Diego, Calif., 1991), all ofwhich are incorporated herein by reference.

The recombinant glycoproteins of the present invention can include anylysosomal enzyme, especially any enzyme useful for enzyme replacementtherapy (ERT). Examples of such enzymes include, without any limitation,acid alpha-glucosidase, acid beta-glucosidase or glucocerebrosidase,alpha-Galactosidase A, acid beta-galactosidase, beta-Hexosaminidase A,beta-Hexosaminidase B, acid sphingomyelinase, galactocerebrosidase, acidceramidase, arylsulfatase, alpha-L-Iduronidase, Iduronate-2-sulfatase,heparan N-sulfatase, alpha-N-Acetylglucosaminidase, Acetyl-CoA,alpha-glucosaminide N-acetyltransferase, N-Acetylglucosamine-6-sulfatesulfatase, N-Acetylgalactosamine-6-sulfate sulfatase, Acidbeta-galactosidase, Arylsulfatase B, acid alpha-mannosidase, acidbeta-mannosidase, acid alpha-L-fucosidase, sialidase, andalpha-N-acetylgalactosaminidase.

In certain exemplary embodiments, the recombinant glycoprotein of thepresent invention is recombinant human β-glucuronidase (rhGUS). Humanβ-glucuronidase is a glycoprotein which contains up to 16oligosaccharides per molecule including a variety of chains that are ofthe high mannose, complex and hybrid types.

Also described herein are isolated or purified glycoproteinpolypeptides. For example, disclosed herein are isolated or purifiedrhGUS polypeptides. The disclosed isolated or purified rhGUSpolypeptides can be used in one or more of the compositions or methodsdisclosed herein.

The rhGUS polypeptides can include the rhGUS peptide sequence as well asfragments thereof, natural variants thereof, and unnatural variantsthereof. The rhGUS sequence is provided in SEQ ID NO: 1. Disclosedherein are isolated or purified polypeptides that consist of SEQ IDNO: 1. Also disclosed herein are isolated or purified polypeptides thatcomprise SEQ ID NO: 1, as well as fragments thereof. Fragments may be atleast about 10, 20, 50, 100, 200, 300, 400, or 500, or more contiguousamino acids. Also disclosed herein are isolated or purifiedpolynucleotides that consist of or comprise a polynucleotide sequencecapable of encoding the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the rhGUS polypeptide has a sialic acid contentgreater than 0.1 mol/mol, 0.5 mol/mol, 0.7 mol/mol, 1 mol/mol, 1.5mol/mol, 2 mol/mol, or 5 mol/mol of the rhGUS polypeptide. In someembodiments, the rhGUS polypeptide has a sialic acid content of about0.5 mol/mol to about 2.0 mol/mol of the rhGUS polypeptide. In oneembodiment, the rhGUS polypeptide has a sialic acid content of about 0.6mol/mol to about 1.5 mol/mol of the rhGUS polypeptide. In anotherembodiment, the rhGUS polypeptide has a sialic acid content of about 0.7mol/mol to about 1.4 mol/mol of the rhGUS polypeptide. In an exemplaryembodiment, the rhGUS polypeptide has a sialic acid content of about 0.8mol/mol to about 1.3 mol/mol of the rhGUS polypeptide. In anotherexemplary embodiment, the rhGUS has a sialic acid content of about 1.0mol/mol to about 1.2 mol/mol of the rhGUS polypeptide.

In additional embodiments, at least 40%, 50%, 60%, 70%, or 80% of thegalactose residues of the rhGUS polypeptide are sialylated. In anexemplary embodiment, at least about 65% to at least about 75% of thegalactose residues of the rhGUS polypeptide are sialylated.

As described herein, the glycoproteins of the invention may be producedrecombinantly. A polynucleotide encoding a recombinant glycoprotein ofthe invention can be introduced into a recombinant expression vector. Inan exemplary embodiment, the recombinant glycoprotein is rhGUS.Accordingly, the application also relates to a recombinant expressionvector comprising a polynucleotide encoding rhGUS. In one embodiment,the rhGUS protein produced by the recombinant expression vector consistsof or comprises SEQ ID NO: 1.

As is understood in the art, recombinant vectors can be expressed in asuitable host cell system using techniques well known in the art.Accordingly, the application also relates to a host cell comprising apolynucleotide encoding rhGUS. In one embodiment, the rhGUS proteinproduced by the host cell consists of or comprises SEQ ID NO: 1.Suitable host cells for expressing the rhGUS protein of the presentinvention can include any cell line that can glycosylate proteins,preferably a mammalian cell line that has been genetically engineered toexpress a protein. For example, Chinese hamster ovary (CHO), HeLa, VERO,BHK, Cos, MDCK, 293, 3T3, myeloma (e.g. NSO, NSI), or WI38 cells may beused. In an exemplary embodiment, the cells used to produce therecombinant glycoprotein are Chinese Hamster Ovary (CHO) cells.

It yet another aspect, the invention provides a formulation comprisingone or more glycoproteins that have a high level or increased sialicacid content. Formulations in general include liquid forms (solutions)such as, but not limited to reconstituted lyophilizates, and solid formssuch as, but not limited to lyophilized forms, gels, microencapsulatedpartilces and pastes. The formulations in accordance with someembodiments of the present invention can be combinations of liquidformulations, lyophilizates, and liquid solitions prepared fromreconstituted lyophilizates used in combination with gel, partiles, orpastes.

In some embodiments, the formulation is a solution including an aqueousbuffer and the recombinant glycoprotein. The buffer may include SodiumPhosphate (Na-Pi), histidine, arginine, glycylglycine, tartaric acid,malic acid, lactic acid, aspartic acid, succinic acid or any combinationthereof. In an exemplary embodiment, the buffer includes Na-Pi andhistidine. In another embodiment, the buffer includes na-Pi, histidineand arginine.

In some embodiments, the buffer further includes one or more otheringedients such as, but not limited to sidium chloride (NaCl),polyxyethylene (Tween-20), potassium chloride, and sorbitol (e.g.,D-sorbitol). In an exemplary embodiment, the buffer includes Na-Pi,histidine, NaCl and Tween 20.

As is well appreciated in the art, the stability of proteins may bedependent upon the pH and/or the ionic strength of a formulation.According to some embodiments of the present invention, the pH of theformulation is about 9.0 to about 5.0, for example, about 7.5 to about6.0. In some embodiments, the pH is about 9.0, about 8.0, about 7.5,about 7.0, about 6.5, about 6.0, about 5.5 or about 5.0. It was thepresent invention that first recognized that lower pH of a formulationwould improve the stability of the recombinant glycoprotein. Forexample, Table 6 in Example 2 demonstrates the improved stability asmeasured by the percentage of tetramers when the pH was changed to 6.0from 7.5.

Methods of Production

In yet another aspect, the present invention provides a method forincreasing the sialylation of a glycoprotein and additionally the M6Plevel of a glycoprotein produced by a cell culture with a serum orprotein free media.

In general, culture media can be divided into several subsets based onthe level of defined media. For example, a culture media can be: 1)Serum-containing media (commonly 10-20% Fetal Bovine Serum (FBS)); 2)Reduced-serum media (commonly 1-5% FBS); 3) Serum-free media (synonymouswith defined media); 4) Protein-free media (no protein but containsundefined peptides from plant hydrolysates); 5) Chemically-defined media(with only recombinant proteins and/or hormones); 6) Protein-free,chemically defined media (contains only low molecular weightconstituents, but can contain synthetic peptides/hormones); and 7)Peptide-free, protein-free chemically defined media (contains only lowmolecular weight constituents).

In some embodiments of the present invention, a reduced-serum media maybe used to grow cells for the expression of glycoproteins. In someembodiments of the present invention, a serum-free media may be used togrow cells for the expression of glycoproteins. In some embodiments, aprotein-free media may be used to grow cells for the expression ofglycoproteins. In some embodiments, a chemically defined media may beused to grow cells for the expression of glycoproteins. In someembodiments, a protein-free, chemically defined media may be used togrow cells as demonstrated in Example 1. Further in some otherembodiments, a peptide-free, protein-free chemically defined media isused to grow cells for the expression of glycoproteins.

As is well understood in the art, serum-free media may contain undefinedanimal-derived products such as serum albumin (purified from blood),hydrolysates, growth factors, hormones, carrier proteins, and attachmentfactors. These undefined animal-derived products will contain complexcontaminants, such as the lipid content of albumin. In contrast,chemically defined media is defined as all of the components beingidentified and having their exact concentrations known. In someembodiments, a chemically defined medium is entirely free ofanimal-derived components. In some embodiments, a chemically definedmedium excludes FBS, bovine serum albumin (BSA), human serum albumin(HAS) or combinations thereof. To achieve this, chemically defined mediais commonly supplemented with recombinant versions of albumin and growthfactors, usually derived from rice or E. coli, or synthetic chemicalsuch as the polymer polyvinyl alcohol which can reproduce some of thefunctions of BSA/HSA.

In some embodiments, the protein free medium described herein does notcontain any proteins or components of biological origin. The absence ofproteins in the medium eliminates the risk from contamination with bloodborne or other pathogens or non-human proteins. In addition, suchprotein free media are usually completely defined as to identity andquantity of all of its ingredients, which may provide unrivalled productconsistency, superior product quality control profile and better productstability than protein-containing media.

In some embodiments, the medium used herein does not include aneffective amount of a sugar selected from galactose, fructose,n-acetyl-mannosamine, mannose and combinations thereof. For example, theeffective amount of a sugar is greater than 0.01 mM, 0.05 mM, or 0.1 mM.

In some embodiments, the present invention provides a method forculturing mammalian cells comprising growing in culture a mammalian cellto produce a protein, e.g., a glycoprotein, in a serum or protein freemedia. Suitable cells for practicing the present invention include anycell line that can glycosylate proteins, preferably a mammalian cellline that has been genetically engineered to express a protein. In someembodiments, cells are homogenous cell lines. Numerous suitable celllines are known in the art. For example, Chinese hamster ovary (CHO),HeLa, VERO, BHK, Cos, MDCK, 293, 3T3, myeloma (e.g. NSO, NSI), or WI38cells may be used. In an exemplary embodiment, the cells used to producethe recombinant glycoprotein are Chinese Hamster Ovary (CHO) cells.

In accordance with some embodiments of the present invention,particularly useful cells are CHO cells, which are widely used for theproduction of recombinant proteins, e.g. cytokines, clotting factors,and antibodies (Brasel et al. (1996), Blood 88: 2004-2012; Kaufman etal. (1988), J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J MolEndocrinol 6: 231-239; Wood et al. (1990), J. Immunol 145: 3011-3016). Adihydrofolate reductase (DHFR)-deficient mutant cell line (Urlaub et al.(1980), Proc. Natl. Acad. Sci. USA 77:4216-4220), such as DXB11 orDG-44, is useful because the efficient DHFR selectable and amplifiablegene expression system allows high level recombinant protein expressionin these cells (Kaufman (1990), Meth. Enzymol. 185: 527-566). Inaddition, these cells are easy to manipulate as adherent or suspensioncultures and exhibit relatively good genetic stability. CHO cells andrecombinant proteins expressed in them have been extensivelycharacterized and have been approved for use in clinical commercialmanufacturing by regulatory agencies.

In some embodiments, cells are grown in a fed batch mode. A fed-batchprocess is defined as an operational technique where one or morenutrients (substrates) are added to a culture medium to increase growthand achieve a high cell density in a bioreactor. Generally, addingnutrients in a controlled manner has a positive effect on the culture'sgrowth rate and production. In some embodiments, a cell concentrationgreater than 10⁶ cells/mL, 10⁷ cells/mL, 2×10⁷ cells/mL, 5×10⁷ cells/mL,or 10⁸ cells/mL in bioreactors can be achieved. In some embodiments,bioreactors used in the fed batch mode have a volume of at least 10 L,20 L, 50 L, 80 L, 100 L, 250 L, 500 L or 1000 L. Cells can be growneither in suspension or adherent cultures. In an exemplary embodiment,cells are grown in suspension. Mammalian cells are preferred, and in aparticular exemplary embodiment, the mammalian cells are Chinese hamsterovary cells.

Therapeutic Treatment

In yet another aspect, the invention provides methods of treating acondition or disorder comprising administering to an individual in needof such treatment a therapeutically effective amount of thecomposition/preparation as described herein.

Compositions/preparations as described herein can be used alone ortogether with any therapeutic agents/compositions for various purposes,such as in the treatment methods described herein. In this regard, thecompositions/preparations can be pharmaceutically acceptable.

In some embodiments, the condition or disorder requiring treatment isassociated with an enzyme deficiency. Enzyme deficiencies in cellularcompartments such as the golgi, the endoplasmic reticulum, and thelysosome cause a wide variety of human diseases. For example, lysylhydroxylase, an enzyme normally in the lumen of the endoplasmicreticulum, is required for proper processing of collagen; absence of theenzyme causes Ehlers-Danlos syndrome type VI, a serious connectivetissue disorder. GnT II, normally found in the golgi, is required fornormal glycosylation of proteins; absence of GnT II leads to defects inbrain development.

In an exemplary embodiment, the condition or disorder associated with anenzyme deficiency is a lysosomal storage disorder (LSD). More than fortylysosomal storage diseases (LSDs) are caused, directly or indirectly, bythe absence of one or more proteins in the lysosome. LSDs arise fromabnormal metabolism of various substrates, including glycosphingolipids,glycogen, mucopolysaccharides and glycoproteins. The metabolism of thesubstrates normally occurs in the lysosome and the process is regulatedin a stepwise process by various degradative enzymes. Therefore, adeficiency in any one enzyme activity can perturb the entire process andresult in the accumulation of particular substrates. Listed below are anumber of lysosomal storage disorders and the corresponding defectiveenzymes:

Pompe disease: Acid alpha-glucosidase Gaucher disease: Acidbeta-glucosidase or glucocerebrosidase Fabry disease:alpha-Galactosidase A GMI-gangliosidosis: Acid beta-galactosidaseTay-Sachs disease: beta-Hexosaminidase A Sandhoff disease:beta-Hexosaminidase B Niemann-Pick disease: Acid sphingomyelinase Krabbedisease: Galactocerebrosidase Farber disease: Acid ceramidaseMetachromatic leukodystrophy: Arylsulfatase A Hurler-Scheie disease:alpha-L-Iduronidase Hunter disease: Iduronate-2-sulfatase Sanfilippodisease A: Heparan N-sulfatase Sanfilippo disease B:alpha-N-Acetylglucosaminidase Sanfilippo disease C: Acetyl-CoA:alpha-glucosaminide N-acetyltransferase Sanfilippo disease D:N-Acetylglucosamine-6-sulfate sulfatase Morquio disease A:N-Acetylgalactosamine-6-sulfate sulfatase Morquio disease B: Acidbeta-galactosidase Maroteaux-Lamy disease: Arylsulfatase B Sly disease:beta-Glucuronidase alpha-Mannosidosis: Acid alpha-mannosidasebeta-Mannosidosis: Acid beta-mannosidase Fucosidosis: Acidalpha-L-fucosidase Sialidosis: Sialidase Schindler-Kanzaki disease:alpha-N-acetylgalactosaminidase

In certain exemplary embodiments, the present invention provides amethod of treating a LSD comprising administering to an individual inneed of such treatment a therapeutically effective amount of thecomposition/preparation as described herein. In one exemplaryembodiment, the composition/preparation comprises recombinant humanβ-glucuronidase. In another exemplary embodiment, the LSD ismucopolysaccharidosis type 7 (i.e., MPS 7, MPS VII, or Sly Syndrome), adisorder resulting from the deficiency of β-glucuronidase. In someembodiments, the recombinant human β-glucuronidase harbors an increasedsialic acid content and is particularly useful in treating a LSD, e.g.,MPS 7.

In some embodiments, the present invention provides a method fortreating a condition or disorder in a subject, comprising administeringa regimen of a composition/preparation as described herein, wherein theadministration provides a statistically significant therapeutic effectfor the treatment of the condition or disorder. In some embodiments, thesubject is human. In some embodiments, the composition/preparationcomprises a recombinant glycoprotein that harbors an increased sialicacid content. In an exemplary embodiment, the recombinant glycoproteinhas a sialylation content of at least 0.7 mol/mol of the recombinantglycoprotein. In another exemplary embodiment, the recombinantglycoprotein has a sialylation content of at least 1 mol/mol of therecombinant glycoprotein. In some embodiments, the condition or disorderis associated with an enzyme deficiency. In an exemplary embodiment, thecondition or disorder associated with an enzyme deficiency is alysosomal storage disorder (LSD).

Accordingly, the present invention provides a method for treating alysosomal storage disorder (LSD) in a subject, comprising administeringa regimen of the composition/preparation as described herein, whereinthe administration provides a statistically significant therapeuticeffect for the treatment of the LSD. In an exemplary embodiment, thecomposition/preparation comprises a recombinant human β-glucuronidasethat harbors an increased sialic acid content. In a further exemplaryembodiment, the LSD is mucopolysaccharidosis type 7 (i.e., MPS 7, MPSVII, or Sly Syndrome).

According to the present invention, treatment of the LSD includes anyform of treating the LSD, e.g., reducing any symptom of the LSD,reducing the severity of any symptom of the LSD, shortening the durationof one or more symptoms of the LSD, treating or inhibiting any cause orcondition associated with the LSD, or reducing any clinical criteria ormeasurement of the degree or condition of the LSD.

According to the present invention, the recombinant humanβ-glucuronidase of the present invention is administered in a regimenfor the treatment of a LSD. In one embodiment, the LSD is MPS 7. Suchregimen includes dosage per administration, per day, per every twoweeks, as well as number of doses per treatment cycle, or combinationsthereof.

In general, the recombinant human β-glucuronidase (rhGUS) of the presentinvention can be administered at a dosage of from about 0.1 mg to 20 mg,0.2 mg to 15 mg, 0.5 to 12 mg, 1 mg to 10 mg. 1.5 mg to 8 mg, 2 mg to 6mg per kg. In some embodiments, the rhGUS is administered at a dosage ofabout 0.1 mg, 0.2 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg,8 mg, 9 mg, 10 mg, 11 mg, or about 12 mg per kg. In an exemplaryembodiment, the rhGUS is administered at a dosage of about 4 mg per kg.Dosages may be adjusted for the condition of each patient as well asother drugs taken by the patient.

In some embodiments, such dosage is administered hourly, daily, weekly(i.e., QW), every two weeks (i.e., QOW), or monthly.

In some embodiments, rhGUS is administered hourly, about every 1 to 24hours, 1 to 20 hours, 1 to 16 hours, 1 to 12 hours, 1 to 8 hours, 1 to 6hours, 1 to 4 hours, 1 to 2 hours or every hour. In some embodiments,rhGUS is administered about every 2, 3, 4, 5, or 6 hours, or isadministered about every 10 minutes, 15 minutes, 30 minutes, 45 minutesor 60 minutes.

In some embodiments, rhGUS may be administered by continuous infusion.In some embodiments, rhGUS may be administered to the patient fortreatment periods of at least about 2, 4, 6, 10, 12 hours, or longer,which may improve effectiveness in some embodiments. In someembodiments, rhGUS is administered by continuous infusion for 1 to 24hours, 1 to 20 hours, 1 to 16 hours, 1 to 12 hours 1 to 10 hours, 1 to 8hours, 1 to 6 hours, 1 to 4 hours to 1 to 2 hours. In some embodiments,rhGUS is administered by continues infusions for about 10 minutes, 15minutes, 30 minutes, 45 minutes or 60 minutes. In some embodiments,rhGUS is administered by continuous infusion for about 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12hours, 24 hours or more. In an exemplary embodiment, rhGUS isadministered by continuous infusion for about 4 hours. In someembodiments, the continuous infusion periods are separated by periods ofnon-infusion (i.e., periods where no rhGUS is administered). Theinfusion may be carried out by any suitable means, such as by minipump.

In some embodiments, rhGUS is administered about every 1 to 30 days,every 1 to 25 days, every 1 to 20 days, every 1 to 14 days, every 1 to10 days, every 1 to 5 days or daily.

In some embodiments, rhGUS is administered for about 1 to 12 weeks,about 1 to 24 weeks, about 1 to 36 weeks, about 1 to 48 weeks, about 1to 60 weeks, or about 1 to 72 weeks. In some embodiments, the rhGUS isadministered for about 1 month, 4 months, 8 months, 12 months, 16months, 20 months, or more. In some embodiments, the rhGUS isadministered for about 1 year, 2 years, 5 years, 10 years, or more. Insome embodiments, the rhGUS is administered permanently (i.e., long-termuse).

The rhGUS may be provided in lyophilized form, and reconstituted withsterile (e.g., aqueous) diluent prior to administration. The rhGUS maybe administered by any effective route, including by subcutaneousinjection, intramuscular injection, intravenous injection or infusion,and orally. In certain exemplary embodiments, the rhGUS is administeredby intravenous infusion. Generally, the scheduled dose of rhGUS may beadministered as a single dose (e.g., injection), or may be spaced outover the course of 24 hours or less, for example, by continuous infusionor repeated injection of subdose, or the like or as describedextensively herein. In one embodiment, the scheduled dose of rhGUS maybe administered as a single injection or as multiple injections.

In one embodiment, the patient receives approximately every other weekly(i.e., QOW) administration of rhGUS, at a dose between about 0.5 and 12mg (e.g., about 1, 2, 4, 8, or 12 mg) per kg to reduce the severity ofthe LSD. In an exemplary embodiment, the patient receives approximatelyevery other weekly administration of rhGUS at a dose of about 4 mg perkg. The regimen may continue in some embodiments for 12, 24, 36, 48, or60 weeks, or permanently (i.e., long-term use).

According to the present invention, the rhGUS used in methods of thepresent invention can be administered either alone or in combinationwith a standard of care for the LSD, or as part of treatment regimeninvolving the standard of care for the LSD. In some embodiments,patients may be administered prophylactic antihistamine prior to eachinfusion of rhGUS. In additional embodiments, patients may beadministered an antipyretic medication (e.g., ibuprofen oracetaminophen) prior to each infusion of rhGUS.

According to some embodiments of the present invention, administrationof rhGUS provides a statistically significant therapeutic effect. In oneembodiment, the statistically significant therapeutic effect isdetermined based on one or more standards or criteria provided by one ormore regulatory agencies in the United States, e.g., FDA or othercountries. In another embodiment, the statistically significanttherapeutic effect is determined based on results obtained fromregulatory agency approved clinical trial set up and/or procedure.

In some embodiments, the statistically significant therapeutic effect isdetermined based on a randomized, placebo-controlled, blind-start,single-crossover clinical trial set up. In some embodiments, thestatistically significant therapeutic effect is determined based on datafrom a clinical trial design whereby subjects are randomized to one of 4groups, each representing a different treatment sequence at differentpre-defined time points in a blinded manner. In some embodiments, thestatistically significant therapeutic effect is determined based on datafrom a patient population of at least 4, 6, 8, 10, or 12 subjects. In anexemplary embodiment, the statistically significant therapeutic effectis determined based on data from a patient population of 12 subjects.

In some embodiments, the statistically significant therapeutic effect isdetermined based on a study involving 12 subjects randomized 1:1:1:1 toone of four treatment sequence groups to either start treatment with 4mg/kg rhGUS every other week (i.e., QOW), or placebo and cross over to 4mg/kg rhGUS QOW at different, pre-defined time points. In someembodiments, the statistically significant therapeutic effect isdetermined based on a study in subjects dosed with either 4 mg/kg rhGUSor placebo QOW for 48 weeks.

In some embodiments, the statistically significant therapeutic effect isdetermined based on a study wherein rhGUS is administered QOW by slow IVinfusion over a period of approximately 4 hours. In some embodiments,patients are pre-medicated with prophylactic antihistamine (e.g.,cetirizine or loratadine) prior to each infusion of rhGUS.

In some embodiments, the statistically significant therapeutic effect isdetermined by measuring urinary glycosaminoglycan (uGAG) levels as theprimary endpoint. Extensive research conducted over the last 20 years onMPS disorders provides significant relevant scientific data that allowsfor the qualification of uGAG levels as a biomarker that is reasonablylikely to predict clinical benefit. The disease process and mechanism ofaction for the rhGUS in MPS 7 are well understood and data from othersimilar MPS disorders with comparable enzyme replacement therapies(ERTs) have established that uGAG is a direct pathophysiological andreadily measured marker of the MPS disease process and uGAG is areasonable predictor of treatment effect and clinical benefit in MPSdisorders. In an exemplary embodiment, the statistically significanttherapeutic effect is determined based on the determination of uGAGlevels a clinical study involving 12 subjects who have been treated with4 mg/kg rhGUS or placebo QOW over a 48-week period.

In some embodiments, the statistically significant therapeutic effect isdetermined using secondary efficacy measures (i.e., secondary endpoints)such as a multi-domain responder index and an evaluation ofindividualized clinical response.

In some embodiments, the statistically significant therapeutic effect isdetermined using a multi-domain responder index, which combinesindependent multi-domain analyses to assure that the broader basis forefficacy can be assessed without the complexity of trying to constructqualified composite endpoints. In some embodiments, the multi-domainresponder index provides an assessment of rhGUS efficacy across a broadspectrum of clinical characteristics commonly observed in MPS 7patients.

In some embodiments, the statistically significant therapeutic effect isdetermined by evaluating individualized clinical response (ICR). This isa measure of each subject's response to treatment that is selected basedon the relevance of the outcome measure to concerns that thesubject/parent/caregiver has reported, the subject's ability to completeclinical outcome assessment reliably, and the extent of impairment forthat individual. Use of an ICR enables evaluation of the clinicalbenefit of rhGUS by assessing change in a prespecified individualizedclinical outcome that is deemed most relevant for each subject and thendetermining an overall response rate for the study population. In someembodiments, the secondary efficacy measures (i.e., secondary endpoints)may include the evaluation of treatment subjects for signs and symptomsof MPS7 that interfere most with the subject's daily life (i.e.,clinical problem evaluation). In some embodiments, the evaluation mayinclude testing of pulmonary function, testing of walking distance,testing of shoulder flexion range of motion, and testing of fine motorfunction.

In some embodiments, the statistically significant therapeutic effect isdetermined based on data with an alpha value of less than or equal toabout 0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, thestatistically significant therapeutic effect is determined based on datawith a confidence interval greater than or equal to 95%, 96%, 97%, 98%or 99%. In some embodiments, the statistically significant therapeuticeffect is determined based on data with a p value of less than or equalto about 0.05, 0.04, 0.03, 0.02 or 0.01. In some embodiments, thestatistically significant therapeutic effect is determined on approvalof Phase III clinical trial of the compositions and methods provided bythe present invention, e.g., by FDA in the US.

In general, statistical analysis can include any suitable methodpermitted by a regulatory agency, e.g., FDA in the US or China or anyother country. In some embodiments, statistical analysis includesnon-stratified analysis, log-rank analysis, e.g., from Kaplan-Meier,Jacobson-Truax, Gulliken-Lord-Novick, Edwards-Nunnally, Hageman-Arrindeland Hierarchical Linear Modeling (HLM) and Cox regression analysis.

In some embodiments, lysosomal storage biomarkers can be used forpredicting treatment response and/or determining treatment efficacy. Insome embodiments, urinary glycosaminoglycan (uGAG) levels can bemeasured and a reduction in uGAG levels employed as an indicator ofpositive treatment response. In some embodiments, elevated levels of theuGAG biomarker, which later decrease upon administration of rhGUS, ispredictive of treatment response. In some embodiments, this informationcan be employed in determining a treatment regimen (as described herein)for the treatment of a lysosomal storage disorder (e.g., MPS 7) usingrhGUS. As such, the present invention provides methods for determining atreatment regimen which includes detecting a decrease in the level of aLSD biomarker in a biological sample from a subject treated with rhGUSand determining a treatment regimen of the rhGUS based on a decrease inthe level of one or more one or more LSD biomarkers in a biologicalsample. In some embodiments, the LSD biomarker is uGAG. In someembodiments, a decreased or reduced level of uGAG is indicative oftreatment response and/or treatment efficacy of treatment with rhGUS. Insome embodiments, reduction of uGAG levels to a predetermined standardlevel is indicative of better treatment prognosis with rhGUS.

EXAMPLES Example 1 Production and Quantitation of Total Sialic Acid

The recombinant human β-glucuronidase (rhGUS) produced in accordancewith the present invention was labeled GUS CR01. The recombinant proteinis produced from Chinese Hamster Ovary (CHO) cells that have beenengineered to express and secrete the enzyme into the culture mediumusing a bioreactor culture system.

Previous batches of p-glucuronidase (labeled as GUS Lot 43/44) have beenproduced using the same cell line by a process in which the cells aregrown attached to microcarriers in a continuous perfusion system. Cellsare generally expanded in growth media containing Fetal Bovine Serum(FBS). Afterward, FBS is washed out and replaced with media containinghydrolysates and supernatant was harvested in perfusion mode.

In contrast to the previously reported methods, GUS CR01 was produced ina culture system in which the cells are grown in suspension in a fedbatch mode. Another difference is GUS CR01 was cultured only inchemically defined protein free medium as opposed to serum containingmedium used previously.

A method for quantitation of total sialic acid in GUS was developed atthe Rentchler Biotechnologie (RB) Quality Control department forrelease-testing of the GUS drug substance. In this method, sialic acidresidues are released from the rhGUS glycan structures with acidhydrolysis. The released sialic acid is then labeled with OPD(O-phenylenediamine dihydrochloride) and analyzed by Reversed-Phase HPLCanalysis (RP-HPLC). To date, Lot 43/44 and six RB-produced lots of rhGUShave been analyzed at RB for total sialic acid. These results (Table 1)are consistent with the result seen at GlycoSolutions.

TABLE 1 Results for Total Sialic Acid Analysis of GUS Sialic Acid(mol/mol Lot GUS Production Method GUS monomer) Lot 43/44 PreviouslyReported 0.04 PR01 In accordance with the 1.0 present invention PR02 Inaccordance with the 0.7 present invention CR01 In accordance with the1.1 present invention GMP1 In accordance with the 1.2 present inventionGMP2 In accordance with the 1.2 present invention GMP3 In accordancewith the 1.2 present invention

Example 2 Pharmacokinetics in Male Sprague Dawley Rats

In this Example, recombinant human β-glucuronidases produced inaccordance with the present invention were compared to ones produced aspreviously reported in the art. The objective of this study was toevaluate the pharmacokinetics and tissue distribution of recombinanthuman p-glucuronidases administered intravenously as a single two hourinfusion in male Sprague Dawley rats. The rate of infusion was ⅓ of thetotal volume for the first hour followed by ⅔ of the total volume in thesecond hour. This dosing regimen was designed to simulate the dosingregimen to be used in the patients.

Materials and Methods

Test Articles and Infusions

Three test articles were used in this study: 0.9% sodium chloride as thenon-enzyme control, GUS CR01 and GUS Lot 43/44. Complete specificationsfor the test articles can be found in Table 2.

TABLE 2 Test Articles Test Article 1 Name: 0.9% Sodium Chloride forInjection, USP (Saline) Source: Baxter Healthcare (Marion, NC) PhysicalProperties: Clear liquid Identifier/Lot Number: C883827 SterilityStatus: Sterile Storage Conditions: Room temperature Expiration DateApril 2014 Test Article 2 Name: GUS CR01 Source: UltragenyxPharmaceutical Inc. (Novato, CA) Quantity: ~32.5 mL Concentration: 2.0mg/mL GUS Activity units/ml 10.75 Munits/ml Specific Activity 5.35Munits/mg units/mg Physical Properties: Clear liquid Identifier/LotNumber: CR01 Storage Conditions: 2 to 8° C. Expiration Date: Notprovided Test Article 3 Name: GUS Lot 43/44 Source: UltragenyxPharmaceutical Inc. (Novato, CA) Quantity: ~25 mL Concentration: 2.5mg/mL* (2.18 mg/ml) GUS Activity units/ml 11.4 Munits/ml SpecificActivity 5.23 Munits/mg units/mg Physical Properties: Clear liquidIdentifier/Lot Number: 43/44 Storage Conditions: −60 to −80° C.Expiration Date: Not provided

The test articles were infused into male Sprague-Dawley rats at a doseof ˜2 mg/Kg body weight during a single infusion consisting of two 1hour phases. One third of the dose was infused over the first hour andtwo thirds of the dose was infused during the second hour (Table 3).

TABLE 3 Rat Group Numbers, Dose and Infusion Rates Dose Dose TotalActual Concen- rate Dose Dose* Test Dose tration (mL/ (mg/ (mg/ Group #Article Gender n Route (mg/mL) min) kg) kg) 1 Saline M 5 iv n/a 1^(st)hr: n/a n/a 0.99 2^(nd) hour: 1.98 2 GUS M 5 iv 0.203 1^(st) hr: ~2¹ ~2¹lot: 0.94 CR01 2^(nd) hour: 1.87 3 GUS M 5 iv 0.253 1^(st) hr: ~2¹  ~1.7¹ lot: 0.78 43/44 2^(nd) hour: 1.57 ¹Dose was based on the averagebody weight of all five rats in each group. *The dose for GUS Lot 43/44was originally based on a protein value of 2.5 mg/ml determined by BCAassay. If the protein concentration was based on absorbance at OD 280and an extinction coefficient of 2.12, the actual dose was 84.8% × 2 =1.7 mg/Kg.

Blood samples were taken from each rat pre-treatment and then atintervals during the slow infusion, fast infusion and post infusionphases by the schedule outlined in Table 4. Blood was allowed to clot,serum was separated and stored frozen at −80° C. pending shipment on dryice for analysis.

TABLE 4 Infusion and Bleed Schedule Stage Nominal Interval Predose SlowInfusion 2 min post start of infusion 10 min post start of infusion 30min post start of infusion 60 min post start of infusion Fast Infusion120 min post start of infusion Post Infusion 2 min (post end ofinfusion) 10 min (post end of infusion) 30 min (post end of infusion) 60min (post end of infusion) 120 min (post end of infusion) 240 min (postend of infusion) 480 min (post end of infusion) 24 hr (post end ofinfusion)

GUS Activity in Serum

β-glucuronidase activity was determined as follows: 25 μL of serumdiluted 1:4 to 1:300 in 0.1 M sodium acetate, pH 4.8, and 1 mg/mLcrystalline BSA was mixed with 50 μL of 10 mM 4-MU-β-D-glucuronidesubstrate in 0.1 M sodium acetate, pH 4.8, 1 mg/mL crystalline BSA. Allsolutions were pre-warmed to 37° C. mixed then incubated at 37° C. for30 minutes. The assays were stopped by the addition of 200 μL glycinecarbonate, pH 10.5, and read on a Molecular Devices M2° plate reader atexcitation/emission wavelengths of 366/446 nm. Activity was expressed as1 unit=1 nmole 4MU released/mL/hr at 37° C.

GUS Activity in Tissues

Tissues were collected at necropsy and placed in cryovials, snap frozenin liquid nitrogen, and stored at −80° C. pending shipment on dry icefor analysis. The distribution of GUS activity in the tissues wasassessed as follows. Whole or partial tissue specimens were thawed andcombined with 10 to 20 volumes of 25 mM Tris, 140 mM NaCl, 1 mMphenylmethyl sulfonyl fluoride, pH 7.2. Tissue homogenates were preparedusing a Kinematica Polytron homogenizer for 30 seconds on ice; theresultant homogenates were freeze/thawed once (at −80° C.) followed bysonication for 20 seconds with cooling on ice. A 25 μL total volume ofeach homogenate was assayed for β-glucuronidase using 4MU-β-glucuronideas described previously. Protein concentration of the homogenates wasdetermined by the bicinchoninic acid method. Tissue β-glucuronidaselevels were expressed as nmoles of 4MU hydrolyzed/hr/mg protein.

Results and Discussion

Pharmacokinetics of GUS CR01 vs. GUS Lot 43/44 in the Plasma

FIG. 1 shows the β-glucuronidase activity in the sera of rats from eachinfusion group during the slow infusion stage, the fast infusion stageand the post infusion stage. The curve for Group 1 infused with salineonly, indicates the low endogenous level of rat β-glucuronidase that ispresent in the sera of these rats. The endogenous level has beensubtracted from the values in the other two plots from the rats infusedwith enzyme.

The plots for both GUS CR01 and GUS Lot 43/44 show a time dependentincrease in enzyme activity levels that reach a steady-state level bythe end of the slow infusion period then increases again concomitantwith the start of the fast infusion period. However in contrast, GUSCR01 reaches a level in the serum 2-fold higher at the end of the slowinfusion and 3-fold higher at the end of the fast infusion periodcompared to GUS Lot 43/44. It can also be seen in FIG. 1 that the rapidclearance of both enzymes from the serum after the infusions cease whichis characteristic for lysosomal enzymes in general.

In FIG. 2, we present the post infusion clearance phase for both enzymesfrom which were calculated the t_(1/2) values. GUS Lot 43/44 is clearedfrom the circulation with a 1^(st) phase t_(1/2) of 4.50 minutes. Incontrast, GUS CR01 is cleared at the slightly slower t_(1/2) of 5.30minutes. Raw clearance data was analyzed by a different method tore-calculate the t_(1/2) values (FIGS. 4A and B). The Cmax (14800 forGUS CR01 vs 4300 for GUS lot 43/44) and the AUC-t (18700 vs 5580) areshowing also a 3 fold higher for GUS (Table 5). The t_(1/2) wascalculated very differently as only the second phase was taken intoaccount. For GUS CR01, the 2^(nd) phase t_(1/2) is 1.1 hr and 0.967 hrfor GUS lot 43/44 (Table 5).

TABLE 5 Clearance Vz Cmax Tmax AUC0-t AUC0-inf Half-life (min*Units/mL)/(Units/mL)/ Group Animal (Units/mL) (h) (h*Units/mL) (h*Units/mL) (h)(mg/kg) (mg/kg) Grp 2-6  12900 0 15300 15500 2.05 2.19E−06 0.00036926-10 CR01 2-7  14200 0 16900 16900 0.794 2.01E−06 0.000138 2-8  14200 018300 18400 0.926 1.84E−06 0.000148 2-9  16700 0 22800 23000 1.031.47E−06 0.000132 2-10 16000 0 20300 20300 0.67 1.66E−06 9.65E−05 Mean14800 0 18700 18800 1.1 1.84E−06 0.000181 CV % 10.4 15.7 15.7 50.5 15.365.4 Geometric 14800 18500 18600 1.01 1.62E−06 0.000159 Mean Grp 3-114640 0 5380 5380 0.677 6.26E−06 0.000367 311-15 43/44 3-12 7010 0.036940 6950 0.832 4.64E−06 0.000349 3-13 4670 0 6220 6250 1.02 5.38E−060.000476 3-14 2740 0.03 5420 5430 1.39 6.20E−06 0.000747 3-15 2270 03970 3980 0.913 5.45E−06 0.000668 Mean 4300 0.0133 5580 5600 0.9676.23E−06 0.000521 CV % 44 136.9 19.9 19.8 27.8 22.1 34.4 Geometric 39705490 5510 0.939 6.11E−06 0.000497 Mean

Tissue Distribution of GUS CR01 vs. GUS Lot 43/44

In addition to clearance of the two enzymes, we assessed the tissuedistribution of GUS CR01 compared to GUS Lot 43/44 in liver, spleen,heart, kidney, brain and lung. Tissue extracts prepared from each ofthese tissues were assayed for β-glucuronidase and protein as describedin the methods. The results of the assays were expressed as units ofβ-glucuronidase activity/mg of tissue protein. The summary of theseassays can be seen in FIG. 4. In each of the graphs of this figure, thetotal enzyme levels including the endogenous rat β-glucuronidase isshown on the left side. On the right side of each graph the averageendogenous enzyme level has been subtracted from the total enzyme level.The average endogenous β-glucuronidase level from each tissue wascalculated using values from all five rats from saline infused Group 1.

In each tissue, the level of GUS in rats infused with either GUS CR01 orGUS Lot 43/44 is higher than the saline infused rats. When theendogenous GUS levels are subtracted, it becomes apparent that ratsinfused with GUS CR01 contain GUS levels that are at least two timesgreater than rats infused with GUS Lot 43/44.

This study was designed to assess the β-glucuronidase pharmacokineticand tissue distribution properties of GUS CR01 and GUS Lot 43/44. Thecurrent study determined that GUS CR01 was cleared from the circulationwith a 1^(st) phase t_(1/2) of 5.30 minutes, compared with a fastert_(1/2) of 4.50 minutes for GUS Lot 43/44. The second phase t_(1/2) isalso a bit bigger for GUS CR01.

More significant differences between the 2 enzymes were demonstrated forCmax, AUC-t and tissue distribution. The maximum concentration (C_(max))of β-glucuronidase activity in the serum at the end of the two hourinfusion period for GUS CR01 was 14,829 units/ml, 4.2 times theconcentration of 3537 units/ml attained by GUS Lot 43/44. This increasein C_(max) could be explained by the accumulation of the slower clearingenzyme to a higher concentration in the blood during the infusionperiod. AUC-t was also highly increased (more than 3 time) with GUS CR01vs. GUS Lot 43/44 as represented in FIG. 1.

Last but not least, we observed that in all of the tissues tested thatGUS CR01 was delivered to tissues at levels up to two times or greaterthan that of GUS Lot 43/44. Changing the clearance characteristics of alysosomal enzyme from the circulation is known to have an effect ontissue distribution. It is conceivable that slowing the t_(1/2) of GUSCR01 in the circulation could allow for a more efficient distribution ofthe enzyme to selected tissues. More importantly the changes in tissuedistribution seem to correlate very well with the changes of Cmax andAUC-t. As can be seen in FIG. 3, the error bars are quite large,reflecting a large range of values obtained in the individual rats ofeach study group. Repeat β-glucuronidase assays in triplicate onselected tissues confirmed the original values leading us to believethat the wide range of values seen in these rats were real.

Previously, analysis of GUS CR01 has shown that whereas the level ofmannose 6 phosphate and most other properties are quite similar betweenthe 2 enzymes, the sialic acid content of GUS CR01 is 28 times that ofGUS Lot 43/44 (1.1 mol/mol GUS monomer vs. 0.04 mol/mol GUS monomer).See Table 6.

TABLE 6 Characteristics of GUS CR01 and GUS Lot 43/44 Characteristic/GUS GUS Lot Assay Units CR01 43/44 GMP1 GMP2 GMP3* Titer mg/L ~400 ~50~400 ~400 ~400 pH −log [H⁺] 7.4 7.5 7.4 7.6 6.0 Purity (Reducing %99.2 >95.0 99.2 98.8 99.3 SDS-PAGE) Tetramer % 97.7 99.0 98.4 98.6 99.1(SE-HPLC) Molecular Weight Daltons 290,249 300,000 300,000 300,000300,000 Tetramer Mass Extinction (mg/mL)⁻¹cm⁻¹ 2.08 — 2.0 2.0 2.1Coefficient Charge pH Range comparable 6.6-7.7 comparable comparablecomparable Heterogeneity (IEF) M6P N-Glycan mol-% 14.2 comparable 14 1412 Analysis (Sum of Peaks 15-17) Sialic Acid (moles/mole 1.1 0.04 1.21.2 1.2 Content monomer) Specific Activity (MU/mg) 3.6 3.70 3.9 3.7 3.5Cellular Uptake Kuptake nM 1.2-1.7 1.4 1.8 1.6 1.4 Half-life in MPS7Days 0-21 d 20.0 18.9 NA NA NA Fibroblasts 5-21 d 21.6 20.5*Monosaccharide analysis indicated that 71% of the galactose residues onGMP3 GUS are sialylated.

By putting together the data it is becoming quite apparent that thebetter tissue distribution of GUS CR01 demonstrated here is due to itsincrease in sialic acid content as sialic acid is well known to slowglycoproteins clearance from the circulation by mannose receptorslocated in the endothelial cells in the interior walls of the bloodvessels. The combination of high sialic acid levels and high affinitymannose-6-phosphate moieties provides an optimal combination forreducing tissue uptake via other carbohydrate receptors due to the highsialic acid content, assuring higher concentrations in circulation andthen achieving excellent tissue uptake in the target tissues due to thehigh affinity mannose-6-phosphate levels.

EXAMPLE 3 Treatment of MPS VII Using Enzyme Replacement Therapy

The purpose of this example is to demonstrate that enzyme replacementtherapy for mucopolysaccharidosis type VII (i.e., MPS VII; Sly'sSyndrome) using recombinantly produced human β-glucuronidase (rhGUS)reduces lysosomal storage in a 36-week clinical study.

In this example, three subjects diagnosed with MPS VII were administeredrhGUS with increased sialic acid content. Dosing was performed accordingto the following 36-week schedule:

Weeks 1-12: 2 mg/kg every other week; Weeks 13-20: 1 mg/kg every otherweek; Weeks 21-28: 4 mg/kg every other week; and Weeks 29-36: 2 mg/kgevery other week.

The safety and efficacy of rhGUS was assessed during the 36-weektreatment schedule. The rhGUS compound appeared to be safe and welltolerated. Importantly, no serious adverse events were observed up to 36weeks and there were no drug-related or hypersensitivityinfusion-associated reactions in any of the three subjects.

To measure efficacy, urinary and serum levels of glycosaminoglycans(GAGs) were first evaluated, as lysosomal accumulation of GAGs is ahallmark of GUS deficiency. A rapid and sustained dose-dependentreduction in urinary glycosaminoglycan (uGAG) was observed in subjectstreated with rhGUS. See FIG. 4. The mean reduction in uGAG at the end ofeach dosing interval is shown in FIG. 5 and illustrates that a 4 mg/kgdose resulted in the greatest reduction of uGAG levels.

A progressive reduction in serum glycosaminoglycan (GAG) was also seenin all three subjects treated with rhGUS. Notably, each subjectdemonstrated at least a 25% reduction in serum GAG levels at the end ofthe 36-week treatment schedule. See FIG. 6.

Lastly, liver size was evaluated in subjects treated with rhGUS, asenlarged liver size is frequently observed in patients suffering fromMPS VII. There was a significant reduction in hepatomegaly resultingfrom the 36-week treatment protocol. See FIG. 7.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the present application belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present application,representative methods and materials are herein described.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties. In the caseof any conflict between a cited reference and this specification, thespecification shall control. In describing embodiments of the presentapplication, specific terminology is employed for the sake of clarity.However, the invention is not intended to be limited to the specificterminology so selected. Nothing in this specification should beconsidered as limiting the scope of the present invention. All examplespresented are representative and non-limiting. The above-describedembodiments may be modified or varied, without departing from theinvention, as appreciated by those skilled in the art in light of theabove teachings.

1.-27. (canceled)
 28. A method for reducing urinary glycosaminoglycan (uGAG) levels in a subject having a lysosomal storage disorder (LSD), comprising administering to the subject a composition comprising a recombinant β-glucuronidase having a sialylation content of at least 0.7 mol/mol.
 29. The method of claim 28, wherein the subject is a human.
 30. The method of claim 28, wherein the recombinant β-glucuronidase is administered at a dose of at least about 0.5 mg/kg.
 31. The method of claim 28, wherein the recombinant β-glucuronidase is administered at a dose of at about 1 mg/kg to about 8 mg/kg.
 32. The method of claim 28, wherein the recombinant β-glucuronidase is administered at a dose of at about 2 mg/kg to about 6 mg/kg.
 33. The method of claim 28, wherein the recombinant β-glucuronidase is administered at a dose of at about 4 mg/kg.
 34. The method of claim 28, wherein the recombinant β-glucuronidase is administered weekly.
 35. The method of claim 28, wherein the recombinant β-glucuronidase is administered every other week.
 36. The method of claim 28, wherein the recombinant β-glucuronidase is administered intravenously.
 37. The method of claim 28, wherein the recombinant β-glucuronidase is administered by continuous infusion.
 38. The method of claim 28, wherein the recombinant β-glucuronidase is administered concurrently with or following antihistamine therapy.
 39. The method of claim 28, wherein the recombinant β-glucuronidase comprises SEQ ID NO:
 1. 40. The method of claim 28, wherein the recombinant β-glucuronidase consists of SEQ ID NO:
 1. 41. The method of claim 28, wherein the recombinant β-glucuronidase has a sialylation content of about 0.8 mol/mol.
 42. The method of claim 28, wherein the recombinant β-glucuronidase has a sialylation content of about 1.0 mol/mol.
 43. The method of claim 28, wherein the recombinant β-glucuronidase has a sialylation content of about 1.2 mol/mol.
 44. The method of claim 28, wherein the recombinant β-glucuronidase has a sialylation content of about 1.0 mol/mol to about 1.2 mol/mol.
 45. The method of claim 28, wherein the LSD is mucopolysaccharidosis type
 7. 